Ion-exchange is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. In principle, ion exchange functions by retaining a target analyte molecules in a stationary phase based on reversible ionic interactions. Typically, the ion exchange composition comprises a stationary phase bearing a capture molecule. Under appropriate buffer and ionic conditions, the capture molecule bears a net charge, the target molecule bears an opposite net charge, while contaminants bear a net neutral charge or a similar net charge to the capture molecule. As a result, the target molecule is retained by the ion exchange composition, while contaminants are removed. The target molecule can then be eluted by neutralizing the charge of the capture or target molecule (e.g. by altering the pH) or treating the composition with another molecule that displaces the target molecule (e.g. by treating with a high salt solution). Such processes are broadly categorized as either cation exchange or anion exchange, depending upon the net charge of the molecule that is being isolated.
Anion exchange is commonly used to purify nucleic acids from complex biological solutions. The nucleic acids can be selectively adsorbed because of their negative charge onto anion exchange resins and then eluted. For low molecular weight nucleic acids, elution buffers typically include salts in high concentrations. Simple organic and inorganic anions have lower selectivity coefficients than nucleic acid, but at high concentrations they can displace nucleic acid from the resins. High molecular weight nucleic acid cannot be eluted by salts because of their extremely high selectivity for the resin. In this case, elution is most often performed by raising the pH, since at high pH weak anion exchange groups of the resin lose their charge.
Thus, elution of nucleic acids from ion exchange resin usually is based on an interrelationship of pH and salt concentration. However, these procedures present some difficulties for downstream processing and analysis of the target nucleic acid. Alkali buffers cannot be used for RNA isolation since RNA is degraded in alkaline solutions. DNA is also alkali-sensitive to some extent, most notably, to the deamination of cytosine (conversion of deoxycytosine into deoxyuracil), especially when heated. Storage of alkali buffers is also problematic, as such solutions are often corrosive, toxic, and have poor shelf stability due to absorbance of CO2 from the air. High salt concentrations that are often employed for elution often interfere with downstream enzymatic reactions such as polymerase chain reaction (PCR), ligation reactions, restriction analysis, cDNA generation or isothermal amplification. Therefore, the resulting nucleic acid eluates need to be desalted, which adds additional handling steps to the nucleic acid isolation procedure and may lead to loss of nucleic acids due to the increased number of handling steps or possible contamination. DNA cannot be directly processed enzymatically because most enzymes are completely denatured under alkaline conditions. Thus, high pH elution also requires additional handling steps because it requires neutralization of the eluate, which further dilutes the eluate, increases labor steps, and may not be appropriate for all enzymatic downstream assays.
In addition, the presence of commonly-used anion exchange materials can inhibit downstream analysis of nucleic acids. As such, current protocols require that the nucleic acid be separated from the anion exchange material before the purified nucleic acid can be analyzed. Unfortunately, complete separation of the eluate and the anion exchange material often cannot be achieved, thus reducing both the efficiency and the accuracy of subsequent analyses and amplifications. Moreover, the additional step of eluting and separating the eluate from the anion exchange material hampers the utility of anion exchange in fully automated nucleic acid purification and analysis procedures.
Thus, materials and methods are disclosed for isolating nucleic acids which do not require additional handling steps prior to a further use of the isolated nucleic acids. In particular, the isolated nucleic acids should be directly suitable for any amplification or analysis procedures.